The ability of HIV-1 to utilize the cellular machinery for nuclear entry allows the virus to infect resting CD4+ T cells and tissue macrophages, the two non-dividing cell types believed to play critical roles in HIV-1 transmission, persistence and pathogenesis. However, the precise mechanism and specific requirement of nuclear entry in HIV-1 replication remains unclear. Capsid is the major determinant for HIV-1 infection of non-dividing cells but also dictates the dependence of HIV-1 on TNPO3, a host molecule recently implicated in HIV-1 nuclear entry. Our preliminary studies found a perfect correlation between the dependence of HIV-1 capsid mutants on TNPO3 and their ability to infect non-dividing cells. The hypothesis being tested in this project is that the capsid protein regulates HIV-1 nuclear entry by assisting intracellular virus complexes in hijacking the TNPO3-dependent cellular machinery and this ability is critical for HIV-1 replication in vivo. In Aim 1, we will determine hw the HIV-1 capsid protein determines TNPO3 usage by HIV-1 and how TNPO3 and other host factors promote HIV-1 nuclear entry. We will next define the specific requirement of this TNPO3-dependent nuclear entry for HIV-1 replication in vivo. In Aim 2, we will study a unique case of HIV-1 capsid evolution in patients carrying the HLA-B27. Preliminary data suggests recurrent selection of TNPO3- dependent viruses in these patients. In Aim 3, we will combine our genetic data with a well-studied macaque model of HIV-1 by using simian immunodeficiency virus (SIV) to directly address the importance of nuclear entry in vivo. Here we will generate and utilize TNPO3-independent SIV mutants, which either retain or lose the ability to infect non-dividing cells. Successful completion of this proposed research will help us understand the mechanism by which HIV-1 exploits the nuclear transport machinery to maximize its propagation.